To make 1ul oligo at correct concentration, take 1ul oligo and 9ul water, combine, transfer 1ul of solution to PCR tube

Keep polymerase on ice at all times

39 ul water can be used in place of 38.5ul water

-DLK_062106

PCR Prep (Phusion Kit)

Create PCR mixture

35.5 H2O

10 uL 5x Buffer

1uL dNTP (10mM)

0.5uL Phusions (polymerase)

1uL oligo 1 (10uL)

1uL oligo 2 (10uL)

1uL DNA Template

Run in PCR machine

Phusion polymerase adds at 1kbp/30sec so adjust 72C step accordingly. Also, if using the program 'JEDCOLONY' there is no need for a lysis step if you are PCRing from a miniprep. The 5min 92C step can be eliminated of shortened significantly.